Name: a method for culturing embryonic c. elegans cells
Uploaded: 2013-09-21T20:00:00
Description: Watch this Scientific Journal Video about A Method for Culturing Embryonic C. elegans Cells at JoVE.com

Asterisks (*) indicate new or modified steps as compared to Christensen et al.1
1. Material Setup
<ol>
	<li>The cell culture procedure requires large quantities of eggs isolated from gravid adults. Grow C. elegans on 8P agar plates seeded with NA22 (available through the C. elegans Genetic Consortium - CGC) bacteria to isolate large quantities of eggs. In these plates the amount of peptone used is 8 times the amount that is normally used for NGM plates. ...

C. elegans is a powerful model organism for deciphering the genetic pathways involved in development, behavior and ageing. Its convenience stems primarily from the ease with which it can be genetically manipulated and from its short life cycle. Despite its convenience, C. elegans has its limitations. C. elegans cells are tiny and confined within a pressurized cuticle that limits the application of methods that require direct access to the cells, such as electrophysiological and pharmacological ...

Caenorhabditis elegans (C. elegans) is a powerful model organism for investigating the molecular bases of cellular function, differentiation, and behavior. While its genome, metabolic and biosynthetic pathways are similar to vertebrates', its genetic and molecular tractability are far greater 2. Among its advantages are its size and simple anatomy, its rapid life cycle (3 days at 25 &deg;C), short life-span (2 weeks) and large number of offspring (&gt;200). Due to its hermaphroditic nature and short life cycle, molecular and genetic manipulations are straightforward in C. elegans, including the generation of transgenic animals 3,4 and the application of gene knock-down techniques such as RNA interference 5. C. elegans body and eggshell are transparent. Therefore cells can be easily visualized in both the adult and the embryo using standard microscopy. In the last 40 years, the C. elegans community has created invaluable resources for C. elegans research including a large collection of mutants, knockouts and transgenics, a detailed description of anatomy and development 6,7, including the full reconstruction of the nervous system 8, and a completely sequenced genome which is well annotated and available to the whole community (<a href="http://www.wormbase.com" target="_blank">www.wormbase.com</a>).
Despite the numerous advantages, some experimental approaches have been challenging in C. elegans. These include the ones that require accessibility to the plasma membrane of the cells and isolation of tissues or cell types. Indeed C . elegans tissues are confined within its pressurized hydrostatic skeleton, which is not easily digested by enzymatic treatment or detergents. At the end of 1990s Miriam Goodman and Janet Richmond pioneered methods for electrophysiological recordings of C. elegans neurons and muscle cells in situ 9,10. While these methods gave us important insights into neuronal and muscle function in vivo, they are challenging and low throughput. Alternative methods to study cell function in vivo had been developed, mostly notably in vivo calcium imaging using genetically encoded calcium sensors such as GCamP and cameleon 11-13. These methods though, do not allow the use of pharmacological tools because they are applied on intact living animals.
The first attempt at culturing C. elegans cells in vitro in large scale was made by Laird Bloom during the preparation of his PhD thesis 14. Unfortunately, difficulties encountered with poor adhesion of the cells to the substrate, poor cell differentiation and survival prevented the establishment of this early protocol as a robust cell culture method. In 1995 Edgar and colleagues published a procedure to investigate cell division and morphogenesis by isolation and culture of a single C . elegans embryos 15. Embryonic cells obtained by digestion of the eggshells with a combination of enzymatic treatment and manual dissociation, continued to proliferate, producing up to ~500 cells 15. Subsequently, Leung and coworkers cultured a small numbers of blastomeres to study intestinal morphogenesis. They showed that one in vitro isolated E blastomere produced polarized intestinal cells that created a structure analogous to the intestinal lumen by interacting with each other through apical adherens junctions 16. Buechner and colleagues also reported a similar method for culturing C . elegans embryonic cells in vitro 17.
Based on this early work, Christensen and colleagues developed a robust protocol for culturing embryonic C . elegans cells in vitro 1. They showed that isolated C . elegans cells can differentiate into various cell types and maintain the features that they possess in vivo, including the expression of cell-specific markers. Several techniques that are challenging in vivo, can be applied on isolated C. elegans embryonic cells. These include electrophysiological 1,18 19, imaging, and immunochemical techniques 20,21, as well as isolation of specific cell types by Fluorescent-Activated Cell Sorting (FACS) for the construction of cell-specific cDNA libraries 22,23. Gene knockdown techniques such as RNA interference (RNAi) can be applied on cultured C. elegans cells 1 and a novel metabolic labeling method using Azido-sugar as a tool for glycoprotein discovery has been recently developed for in vitro cultured C. elegans cells 24.
In conclusion, the cell culture method expands the array of techniques that can be applied to the C. elegans model in an effort to decipher gene function in the context of a living organism. We describe here the protocol for culturing C. elegans embryonic cells in vitro, which is largely based on the protocol first described by Christiansen and colleagues 1.

<table border="1">
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			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Bacto Peptone</td>
			<td>VWR International Inc.</td>
			<td>90000-382</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Difco Agar Granulated</td>
			<td>VWR International Inc.</td>
			<td>90000-784</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bacto Tryptone</td>
			<td>VWR International Inc.</td>
			<td>90000-284</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bacto Yeast Extract</td>
			<td>VWR International Inc.</td>
			<td>90000-724</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Leibovitz's L-15 Medium (1x) Liquid</td>
			<td>Invitrogen</td>
			<td>11415-064</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>16140-063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>P4333-100ML</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chitinase from Streptomyces Griseus</td>
			<td>Sigma</td>
			<td>C6137-25UN</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NA22 Escherichia coli</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peanut Lectin</td>
			<td>Sigma</td>
			<td>L0881-10MG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>57903-1KG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-(+)Glucose</td>
			<td>Sigma</td>
			<td>67528-1KG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis (2-aminoethylether), N,N,N',N'- tetraacidic acid (EGTA)</td>
			<td>Sigma</td>
			<td>E0396-25G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma</td>
			<td>H3375-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>57653-1KG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266-100G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2643-500 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma</td>
			<td>P2222-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P9791-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma</td>
			<td>P1767-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Autoclaved distilled H2O</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>101-1000 &mu;l Blue Graduated Pipet Tips</td>
			<td>USA Scentific</td>
			<td>1111-2821</td>
			<td>&nbsp;</td>
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			<td>10 ml Sterilized Pipet Individually Wrapped</td>
			<td>USA Scentific</td>
			<td>1071-0810</td>
			<td>&nbsp;</td>
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			<td>Ergonomic Variable Volume (100-1000 &mu;l) Pipettor with tip ejector</td>
			<td>VWR International Inc.</td>
			<td>89079-974</td>
			<td>&nbsp;</td>
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			<td>Portable Pipet Aid, Drummond</td>
			<td>VWR International Inc.</td>
			<td>53498-103</td>
			<td>&nbsp;</td>
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			<td>Transfer Plastic Pipet Sterile</td>
			<td>VWR International Inc.</td>
			<td>14670-114</td>
			<td>&nbsp;</td>
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			<td>15 ml Conical Tube</td>
			<td>USA Scentific</td>
			<td>1475-1611</td>
			<td>&nbsp;</td>
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		<tr>
			<td>50 ml Conical Tube</td>
			<td>USA Scentific</td>
			<td>1500-1811</td>
			<td>&nbsp;</td>
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		<tr>
			<td>Sterile 18 gauge Needles</td>
			<td>Becton, Dickinson and Co.</td>
			<td>305196</td>
			<td>&nbsp;</td>
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		<tr>
			<td>Sterile 10 ml Syringes</td>
			<td>Becton, Dickinson and Co.</td>
			<td>305482</td>
			<td>&nbsp;</td>
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		<tr>
			<td>Plastic Syringe Filters Corning 0,20 &mu;m pore size</td>
			<td>Corning</td>
			<td>431224</td>
			<td>&nbsp;</td>
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			<td>Acrodic 25 mm Syringe filter w/5 &mu;m versapor Membrane</td>
			<td>VWR International Inc.</td>
			<td>28144-095</td>
			<td>&nbsp;</td>
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		<tr>
			<td>60x15 mm Petri Dish Sterile</td>
			<td>VWR International Inc.</td>
			<td>82050-548</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100x15 mm Petri Dish Sterile</td>
			<td>VWR International Inc.</td>
			<td>82050-912</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>12 mm Diameter Glass Coverslips</td>
			<td>VWR International Inc.</td>
			<td>48300-560</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Clear Cell Culture Plates 24 Well Flat Bottom w/lid</td>
			<td>Thomas scientific</td>
			<td>6902A09</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dumont #5- Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge 5702</td>
			<td>Eppendorf</td>
			<td>022629883</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Inverted Microscope with x10 objective</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ambient air humidified Incubator</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a method for culturing embryonic c. elegans cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

C. elegans cultured cells differentiate and express cell specific markers
Christensen and colleagues using trypan blue staining demonstrated that &gt;99% of embryonic C. elegans cells survive the isolation procedure. At day 9 and 22 after plating, 85% and 65%, respectively are still alive 1. Isolated embryonic C. elegans cells must adhere to a substrate in order to differentiate. Cells that fail to adhere form clumps and it is not clear whether they survive. ...

Watch this Scientific Journal Video about A Method for Culturing Embryonic C. elegans Cells at JoVE.com

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

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